Technical Field
This invention relates to nucleic acid hybridization, and more particularly to a method and means for annealing complementary nucleic acid molecules at an accelerated rate.
The use of nucleic acid probes to detect particular target nucleic acid sequences in samples containing one or more nucleic acids is of vast utility to research, medicine, and forensics. Because nucleic acid probes are highly specific for their target sequences, they can be used as diagnostic reagents to detect the presence of a particular nucleic acid, as well as features within that nucleic acid. Commercial nucleic acid probe assays are being developed for the detection of infectious microorganisms, viruses, mutations in the human genome, as well as for fingerprinting human and other species' genomes. Research applications of nucleic acid probes are manifold, having been extensively utilized in recombinant DNA work for over 10 year.
All nucleic acid probe assays require a step in which a labeled or tagged probe nucleic acid is hybridized to a target sequence by annealing of the probe and target nucleic acids. The time required for such hybridization is often a critically limiting factor in nucleic acid probe assays which are performed in a nonresearch or medical setting in which minimization of the time required for performance of the assay is of importance. The rate of the annealing reaction is affected by several factors, such as ionic strength, temperature, concentration of the reactant molecules, and the presence of denaturing solvent. Concentration of the reactant molecules is perhaps the most important of these factors, because it limits the rate at which the random collisions between the complementary single-stranded nucleic acid probe and target sequences occur as required to bring about annealing thereof to each other in a hybridization reaction. Once two complementary nucleic acid molecules have appropriately collided, they rapidly anneal to form a thermodynamically stable duplex that does not spontaneously dissociate into its single-stranded components.
In the simplest of forms, nucleic acid probe hybridization involves the detection of a target nucleic acid (RNA or DNA), either bound to a solid support or free in solution, using a labelled complementary probe nucleic acid. Nucleic acid probe assays fall into two general categories, i.e., free-solution probe assays and filter (or solid support) binding assays.
In the free-solution probe assays the target and probe nuceic acids are freely dissolved in solution. Target nucleic acid (RNA or DNA) is first extracted from the sample, denatured to convert it to single-stranded form, and dissolved in hybridization buffer. Extraction of target nucleic acid from the sample and denaturation thereof can be accomplished by the procedure disclosed by T. Maniatis et al in "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory (1982), pages 191 to 198. A labelled probe DNA or RNA complementary to the target nucleic acid is added to this solution and allowed to hybridize to the target sequence. When the hybridization reaction is complete, a suspension of hydroxyapatite (calcium hydroxide) is added. The hydroxyapatite selectively binds double-stranded probe/target nucleic acid duplexes as well as other double-stranded molecules, but does not bind unannealed single-stranded molecules. The insoluble hydroxyapatite with probe/target sequence duplex bound thereto is separated from the hybridization medium by centrifugation and washed to remove traces of unreacted probe molecules. If the probe has an isotopic label, the amount of probe bound to the hydroxyapatite is quantitated by scintillation counting. Other conventional means can be used to detect and quantitate nonisotopically labeled probe bound to the hydroxyapatite.
Filter binding DNA probe assays can be performed in several different ways. One way involves binding of single-stranded sample nucleic acid (RNA or DNA) to a nitrocellulose or nylon filter in an irreversible manner. This can be accomplished by applying the nucelic acid to the filter, and then baking it at a temperature of 70 C. for from about one to about two hours under reduced pressure, i.e. at a pressure of less than 1 psi. The filter with sample nucleic acid bound thereto, is then prehybridized by placement thereof in an aqueous solution containing salts, protein, nonreactive DNA or RNA, sodium dodecyl sulfate detergent, buffer, EDTA, and formamide to block nonspecific binding sites on its surface. This procedure is disclosed by Grunstein M. et al, in their paper entitled "Colony Hybridization: A Method for the Isolation of Cloned DNAs that Contain a Specific Gene", Proc. Nat'l Acad. Sci. 72(10): 3961-3965, 1975.
When the prehybridization step is complete, labeled probe DNA or RNA, dissolved in an aqueous solvent, is added to the solution containing the prehybridized filter to which the sample nucleic acid is bound. The probe is allowed to hybridize with the filter-bound sample nucleic acid until formation of sample/probe duplexes has gone to completion. The filter is then removed from the hybridizaton solution and washed with a buffered salt solution at a controlled temperature to remove nonspecifically bound labeled probe sequences. After the washing step, only labeled probe molecules which are specifically annealed to matching sample target sequences remain on the filter. The washed filter can be autoradiographed, or other appropriate conventional means can be used to detect the label and determine the amount and location of the bound probe, and thereby the location of the complementary sample sequences originally applied.
The foregoing procedures employ techniques disclosed in the following prior art literature references:
Meinkoth, J and Wahl, J., "Hybridization of Nuleic Acids Immobilized on Solid Supports", Analytical Biochemistry, 138: 267, 1984; and PA0 Thomas, P. S., "Hybridization of Denatured RNA and Small DNA Fragments Transferred to Nitrocellulose", Proc. Nat'l Acad. Sci., 77 (9): 5201-5205, 1980. PA0 Wetnur, J., Biopolymers, 14: 2517-2524, 1975; PA0 Chang, C. T. et al, Biopolymers, 13: 1847-1858, 1975; and PA0 Kohne, D. E., ACPR: 20-29, November 1986.
A variation of the filter-binding procedure is the sandwich filter-binding assay. This procedure is similar to the filter binding assay, except that it involves two nucleic acid probes. The first probe is unlabeled, and before the assay procedure is undertaken, this probe is covalently bound to the nitrocellulose or nylon filter. The thus prederivitized filter, with the first probe bound thereto, is prehybridized in the same kind of buffer solution as was described above, after which a solution containing single-stranded nucleic acid target sequences extracted from the sample as aforedescribed, are added to the prehybridizaton solution containing the filter. Sample nucleic acid target sequences which are complementary to the bound first probe sequences become adherent to the filter by annealing to said first probe sequences.
A second labeled DNA or RNA probe which is complementary to the filter-bound sample target sequences, but is nonoverlapping with the first filter-bound probe, is then annealed to the sample target sequences and thereby also becomes bound to the filter. Each resultant bound sandwich nucleic acid complex contains the first probe bound to the filter, the target sample nucleic acid sequence annealed to the first probe, and the second labeled probe annealed to the overhanging ends of the target nucleic acid sequence. The steps of a sandwich filter binding assay can be carried out sequentially as aforedescribed, or the sample target sequence and labeled secondary probe nucleic acids can be added to the hybridization medium at the same time. This procedure is discussed by Mulcahy, L., in "DNA Probes: An Overview", ACPR: 14-19, November 1986.
It has been found that the rate of DNA or RNA hybridizaton in the free-solution type probe assay can be accelerated by the addition to the hybridization medium of water soluble polymers, such as dextran sulfate, polyvinyl pyrrolidone, or tetraethyl ammonium chloride. The mechanism by which these polymers enhance the rate of hybridization of DNA or RNA molecules is believed to involve a reduction in the effective solvent volume available to the nucleic acids in solution. The negatively charged polymers complex with available solvent molecules from around the nucleic acid molecules, resulting in an effective increase in concentration of DNA or RNA molecules relative to each other. Such concentration is believed to be effective to increase the number of collisions between complementary sequences, and to thereby produce faster annealing rates.
Such rate enhancer compounds have been found to increase nucleic acid probe hybridization rates by 10 to 200 fold in free-solution probe assays, thereby making possible hybridization times of 1 to 2 hours, rather than overnight. In the case of short synthetic nucleic acid probes, hybridization reactions can be completed in less than 15 minutes if high concentrations of oligomeric probe, for example 1 milligram per milliliter, are used along with rate enhancer compounds. In general, however, the hybridization reaction for nucleic acid probe assays requires 1 to 2 hours when probes of 100 or more nucleotides in length are used in free-solution type hybridization assays. In contrast to results produced by these rate enhancer compounds in free-solution type assays, their use in filter binding type assays has been found to produce no significant rate enhancement.
References discussing the use of rate enhancer compounds are:
In order to supply the frequent need of researchers and others to collect a dense amount of nucleic acid molecules, for example on a carrier membrane, instruments are available commercially which can separate nucleic acid from a gel or can isolate or concentrate nucleic acid molecules from a solution thereof. The operation of such electroelution or electrophoretic concentration devices takes advantage of the fact that, due to the presence of phosphate groups on the nucleic acid backbone, DNA and RNA in aqueous solution are highly negatively charged molecules. When a voltage is applied across platinum wire electrodes placed in a solution of RNA or DNA, the resulting current flow through the solution causes the negatively charged nucleic acid molecules to migrate toward the positive electrode (anode) and concentrate on its surface.
In the aforementioned commercial devices, this principle is used to electrophoretically concentrate the migrating DNA or RNA molecules from a solution, or from agarose or acrylamide containing such molecules, onto the surface of a liquid permeable, for example a cellulose, collector membrane which is impermeable to the nucleic acid molecules and is positioned to prevent such molecules from contacting the anode. Usually, devices of this sort are configured with two chambers separated by the membrane. In one chamber the gel or nucleic acid-containing moiety is placed in a buffered solution near but not against one side of the membrane. The second chamber contains only buffered solution in contact with the other side of the membrane so that aqueous solution contacts both sides of the latter. Platinum wire electrodes present in the respective chambers are connected to a constant direct voltage power supply, the electrode in the chamber containing the nucleic acid to be concentrated being connected to the negative terminal of the source to provide a cathode, and the other electrode being connected to the positive terminal thereof to provide an anode. The electric potential impressed across the electrodes by the source, causes current flow therebetween through the aqueous solutions and is effective to cause the negatively charged nucleic acid molecules in the cathode chamber electrophoretically to migrate toward and be concentrated onto the side of the membrane or disc exposed in the cathode chamber. The nucleic acid becomes deposited on the membrane or disc during the procedure. Upon completion of the concentration step, the electrodes are disconnected from the source, and the nucleic acid deposited on the membrane can be easily removed therefrom, as by washing.
Depending upon the type of membrane used therein, the commercial devices can also be used to bind to the membrane the nucleic acids concentrated thereon. For example, when a membrane of modified nylon is used, the nucleic acids concentrated thereon are bound thereto upon contact. On the other hand, when a membrane of nitrocellulose is used, the nucleic acids concentrated thereon can be bound thereto upon removal of the membrane from the instrument. Such binding can be accomplished by baking at a temperature of 70.degree. C. for about one to about two hours at reduced pressure, i.e. less than 1 psi.
Examples of commercial electrophoretic concentration/elution instruments of the type discussed above are the Electro-Eluter/Concentrator available from C. B. S. Scientific, Del Mar, CA 92014; the preparative gel electrophoresis system (prep gel.TM.) available from Bethesda Research Laboratories, Bethesda, Md.; and the Trans-Blot Cell availabe from Bio-Rad, Richmond, CA.